Exposure of cells to ionizing radiation (IR) activates DNA damage responses leading to a comprehensive reprogramming of gene expression by regulation of both transcriptional and post- transcriptional events. The mechanisms regulating transcriptional and post-transcriptional events are not fully elucidated and a better knowledge about these mechanisms could help improve radiation therapy in the clinic. We will in this revised R21 grant proposal use the newly developed BrU-Seq technique to study these mechanisms in detail. In Specific Aim #1, the global effects of IR on the synthesis and stability of all RNAs in human fibroblasts will be explored. Preliminary experiments show that the stress kinase ATM is required for increased synthesis and stability of certain mRNAs such as BTG2 in human fibroblasts and the hypothesis to be tested is that there may be many more genes affected at the level of synthesis and/or stability in an ATM-dependent way following exposure to IR. In Specific Aim #2, the effects of IR on alternative splicing will be interrogated. Preliminry results show that intron retention is common among processed mRNAs in human fibroblasts and our hypothesis is that since a large number of splicing factors are substrates for activated ATM, IR may affect the splicing code signature in human cells in an ATM-dependent manner. In Specific Aim #3 the effects of IR on the activation of transcription start sites (TSS) and enhancer elements in the genome will be studied using a technique involving UV-irradiation prior to the BrU pulse-labeling to introduce random transcription-blocking lesions in the genome. Preliminary results show that with this technique, all transcription start sites and potentially enhancer elements can be mapped in the genome. The hypothesis to be tested is that IR may affect the selection of TSS and enhancer elements and that ATM may be involved in regulating these alterations. The major innovation of this R21 proposal is the use of the novel BrU-Seq technique for global exploration of how IR affects the transcriptome, the RNA stabilome, the splicing code and selection of TSS and enhancer elements. The analysis will not only include mRNAs but also non-coding RNAs such as microRNAs. These studies may have a great impact on not only our understanding of the mechanisms of altered gene and microRNA regulation following exposure to IR but also, this new technique will have general applications for studying mechanisms of gene expression in other settings.